BB ^BPrincipal Investigator/Program Director (Last, first, n^m>: Beckman, Joseph S. DESCRIPTION: State the application's broad, long-term objectives and specific aims, making reference to the^Kdth relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This description is meant to serve as a succinct and accurate description of the proposed work when separatedfrom the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEEDTHE SPACE PROVIDED. Our long-term goal is to understand how mutations to SOD can increase oxidative stress and cause the death of motor neurons in amyotrophic lateral sclerosis (ALS). We have shown that endogenous formation of peroxynitrite by the diffusion-limited reaction between superoxide and nitric oxide induces apoptosis in cultured embryonic rat motor neurons deprived of trophic support. Both inhibitors of nitric oxide synthesis as well as Cu.Zn superoxide dismutase (SOD) delivered intracellularly with liposomes protect motor neurons from apoptosis. These data indicate that the interaction between nitric oxide and superoxide has a role in motor neuron apoptosis. Mutations to SOD are implicated in the selective degeneration of motor neurons in ALS and expression of ALS- SOD mutants in transgenic mice produces motor neuron disease. A common phenotype among the ALS-SOD mutations so far investigated is to decrease the affinity for zinc. We have shown that zinc-deficient SOD is both less efficient at scavenging superoxide and a better catalyst of tyrosine nitration. Furthermore, the copper in zinc-deficient SOD can act as a non-specific one-electron oxidase, robbing electrons from antioxidants like ascorbate and glutathione that can be transferred to oxygen to produce superoxide. In the presence of NO, zinc-deficient SOD can catalyze the formation of peroxynitrite. In the previous cycle of funding, we have shown that zinc-deficient SOD induces apoptosis in motor neurons by a nitric oxide-dependent mechanism. For the renewal, our first aim is to further investigate the mechanisms by which zinc-deficient SODs can kill cultured motor neurons and to determine what can protect motor neurons from this toxicity. Our second aim is to characterize the source or sources of superoxide induced in motor neurons by trophic factor withdrawal. Our third aim is to test the role of tyrosine nitration by peroxynitrite in the death of motor neurons induced by either trophic factor deprivation or by zinc-deficient SOD. Completion of the specific aims will provide a mechanistic basis for explaining how motor neurons are particularly vulnerable to SOD mutations and establish a link between sporadic and familial SODs. PERFORMANCE SITE(S) (organization, city, state) University of Alabama at Birmingham, Birmingham,Alabama KEY PERSONNEL. See instructions on Page 11. Use continuationpages as neededto providethe required information in the format shownbelow. Name Organization Role on Project Joseph S. Beckman,Ph.D. Alvaro G. Estevez, Ph.D. Yingxin Zhuang Andres Kamaid Ahmad Adhami University of Alabamaat Birmingham PrincipalInvestigator University of Alabama at Birmingham Co-Investigator University of Alabama at Birmingham Research Associate University of Alabamaat Birmingham Research Assistant University of Alabamaat Birmingham Research Assistant PHS 398 (Rev.4/98) Page2 BB Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CC Prin^Plnvestigator/Program Director (Last, first, middle): ^F Beckman, Joseph S. Type the name of the Principal Investigator/Program Director at the top of each printed page and each continuation page. (For type specifications see instructions on Page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, and Personnel 2 Table of Contents 3 Detailed Budget for Initial Budget Period Budget for Entire Proposed Period of Support Budgets Pertaining to Consortium/Contractual Arrangements Modular Budget with Justification 4-5 Biographical Sketch-Principal Investigator/Program Director (Not to exceed two pages) 6-8 Other Biographical Sketches (Not to exceed two pages for each) 9-16 Other Support Resources 17 Research Plan Introduction to Revised application (Not to exceed 3 pages) Introduction to Supplemental application (Not to exceed 1page) a. Specific Aims 18 b. Background and Significance 18-22 c. Preliminary Studies/Progress Report (Items a-d: not to exceed25 pages') 23-32 d. Research Design and Methods 32-43 e. Human Subjects 43 f. Vertebrate Animals 43-44 g. Literature Cited 44-51 h. Consortium/Contractual Arrangements i. Consultants Checklist 52 *Type density and type size of the entire application must conform to limits provided in instructions on page 6. Appendix (Five collatedsets. Nopage numberingnecessary for Appendix) Number of publications and manuscripts accepted for publication (not to exceed ten): _5 _J^M I y [unreadable] appendix is Other items M: IX I induded PHS 398 (Rev. 4/98) (Form Page 3) Page 3 CC Number pages consecutively at th4e bottom throughout the application. Do not use suffixes such as 3a, 3b.